Translation of the abstract (English)

The protein Sam68 (src-associated in mitosis, 68 kDa) is involved in many cellular processes like cell-cycle or apoptosis regulation. Furthermore it has been shown to be important for the RNA metabolism of human immunodeficiency virus (HIV), although different mechanism of action are discussed. Sam68 comprises a central RNA-binding domain flanked by unstructured regions, which contain docking ...

Translation of the abstract (English)

The protein Sam68 (src-associated in mitosis, 68 kDa) is involved in many cellular processes like cell-cycle or apoptosis regulation. Furthermore it has been shown to be important for the RNA metabolism of human immunodeficiency virus (HIV), although different mechanism of action are discussed. Sam68 comprises a central RNA-binding domain flanked by unstructured regions, which contain docking sites for signal transduction proteins. Among them are seven proline-rich sequences (P0 to P6) with a PxxP core motif, that serve as potential docking sites for SH3 domains. Although several proteins which bind to Sam68 via SH3 domains have already been described in the literature, a systematic analysis of the SH3 binding profile has not be made so far.
To this end, a phage display procedure applying a library that displays all human SH3 domains has been carried out in the present work. Thereby more than 30 potential Sam68-binders were identified. Among the nine domains with highest binding affinity were six already known ones (Fyn, Lyn, Nck1, p85alpha, Src and Yes) as well as three SH3 domains characterized as binders for the first time (Hck, Intersectin 2, OSF1). Due to the observed breadth of interacting partners it is proposed to consider Sam68 as a scaffold protein. The detailed characterization of these SH3 domains showed that only the Sam68-motifs P0, P3, P4 and P5 serve as docking sites.
To determine if Sam68-SH3-interactions play a role in the context of HIV replication, Sam68 mutants without the relevant SH3-binding sites were constructed (Sam68-Delta-Px). However, overexpression of Sam68-Delta-Px during HIV replication had no influence on particle production. In a functional knock-down system of endogenous Sam68 by the mutant Sam68-Delta-C, the Sam68-Delta-Px mutants proved to be as effective as the wild-type protein in complementing the Sam68-Delta-C defect. Therefore, Sam68-SH3-interactions likely are dispensable for Sam68's function during HIV replication.
In contrast, the expression of the high-affinity SH3-domain Yes and it's variant Yes-opt (60-fold increased affinity) severely impaired particle production in an HIV replication assay. Most likely these isolated SH3 domains bind to Sam68 and disturb complex formation with other important components. Therefore these SH3 domains represent interesting candidates for a potential HIV gene therapy approach, since they target a cellular protein that is essential for the virus. Addition of a nuclear export sequence to the SH3 domains with the aim to change Sam68's subcellular localization also exhibited a potent antiviral effect, which, however, proved to be independent of Sam68.